The long term goal of the proposed research is to understand the molecular globin gene switch during erythroid development. Both the alpha-like and beta-like families of human globin genes are arranged as individual gene cluster, and the transcriptional turning on-and-off of their members are controlled in cis by the so-called locus-control gene (alpha-LCR and beta-LCR) located at many kb upstream of each gene cluster. A research plan is designed to further study the molecular basis of positive and negative regulation of different human globin genes, in particular the two alpha-like ones, embryonic zeta and adult alpha, by the LCRs. In the next five years, well-established molecular biology techniques and erythroid cell culture-DNA transfection systems will be applied to investigate in detail the transcriptional activation of the zeta and alpha globin genes, in erythroid cells of appropriate developmental stages, by the functional and probably physical interaction among multiple nuclear factor-DNA complexes formed at the globin promoters and the LCRs. This analysis is particularly interesting in view of the recent identification of positive regulatory elements in both the alpha- LCR and the zeta globin promoter, and the observation of erythroid lineage-and developmental stage-specific patterns of nuclear factor- binding in vivo in the alpha-LCR and the alpha globin promoter. This analysis could also lead to the identification of negative regulatory elements responsible for repression of embryonic zeta globin in adult erythroid cells. The investigator will also test the possibility that transcriptional repression of the two adult alpha globin genes, which are unmethylated CpG island, in non-erythroid cells, and in embryonic erythroid cells, is caused by a long range DNA methylation effect. Finally, for future investigation of the functional role of protein-protein interaction and protein-nucleosome interaction in human globin gene switch, it is essential to know the identity of nuclear factor(s) binding in vivo to different regulatory sequence motifs in the globin promoters and LCRs. Most of these motifs, however, could bind to one of several different nuclear factors in vitro. Several alternative immunological precipitation approaches will be adopted and refined to solve the above problem. The same approaches will also be used to test whether RNA polymerase II molecules bind, or pause, near inactive globin promoters in erythroid cells of non-expressing developmental stages. If successful, these studies may advance the understanding and pave new ways for further investigation of the molecular basis of human globin switch. The new concepts and technologies derived would also contribute to the field of eukaryotic gene regulation.